The prognosis for metastatic cancer remains highly unfavorable. Despite advances in radiation therapy and chemotherapy, the long term survival of treated patients has shown only marginal improvement over the past few decades. The lack of significant treatment options available for metastatic cancers emphasizes the need to focus on the development of novel therapeutic strategies. In this regard, targeting tumor vasculature of solid tumors has recently shown promising results in several animal model systems (Baillie et al. (1995) Br. J. Cancer 72:257-67; Bicknell, R. (1994) Ann. Oncol. 5 (Suppl.) 4:45-50; Fan et al. (1995) Trends Pharmacol. Sci. 16:57-66, Thorpe, P. E. and Burrows, F. J. (1995) Breast Cancer Res. Treat. 36:237-51; Burrows, F. J. and Thorpe, P. E. (1994) Pharmacol. Ther. 64:155-74). In a nude mouse model, for instance, introduction of a wild type VHL gene into 786-0 cells, a RCC tumor cell line, inhibited tumor growth (Iliopoulos et al. (1995) Nat. Med. 1:822-26) and angiogenesis.
The growth of solid tumors beyond a few mm3 depends on the formation of new blood vessels (Folknan, J. (1971) N. Engl. J. Med. 285:1182-86). Numerous studies have shown that both primary tumor and metastatic growth are angiogenesis-dependent (Folkman, J. (1971) N. Engl. J. Med. 285:1182-86; Folkman, J. (1972) Ann. Surg. 175:409-16; Folknan, J. and Shing, Y. (1992) J. Biol. Chem. 267:10931-34; Folkman, J. (1996) Sci. Am. 275:150-54). A number of angiogenesis inhibitors have been identified. Certain ones, such as platelet factor-4 (Maione et al. (1990) Science 247:77-79; Gupta et al. (1995) Proc. Natl. Acad. Sci. (USA) 92:7799-7803), interferon, interferon-inducible protein-10, and PEX (Angiolillo et al. (1995) J. Exp. Med. 182:155-62; Stricter et al. (1995) Biochem. Biophys. Res. Commun. 210:51-57; Brooks et al. (1998) Cell 92:391-400), are not xe2x80x9cassociated with tumors,xe2x80x9d whereas two others, angiostatin and endostatin, are xe2x80x9ctumor-associatedxe2x80x9d (O""Reilly et al. (1994) Cell 79:315-28; O""Reilly et al. (1997) Cell 88:277-85). Angiostatin, a potent endogenous inhibitor of angiogenesis generated by tumor-infiltrating macrophages that upregulate matrix metalloelastase (Dong et al. (1997) Cell 88:801-10), inhibits the growth of a wide variety of primary and metastatic tumors (Lannutti et al. (1997) Cancer Res. 57:5277-80; O""Reilly et al. (1994) Cold Spring Harb. Symp. Quant. Biol. 59:471-82; O""Reilly, M. S., (1997) Exs. 79:273-94; Sim et al. (1997) Cancer Res. 57:1329-34; Wu et al. (1997) Biochem. Biophys. Res. Commun. 236:651-54). Recently, O""Reilly, et al. ((1997) Cell 88:277-85) have isolated endostatin, an angiogenesis inhibitor from a murine hemangioendothelioma cell line (EOMA). Circulating levels of a fragment of human endostatin have been detected in patients with chronic renal insufficiency with no detectable tumor (Wu et al. (1997) Biochem. Biophys. Res. Commun. 236:651-54).
The amino terminal sequence of endostatin corresponds to the carboxy terminal portion of collagen XVIII. Endostatin is a specific inhibitor of endothelial proliferation and angiogenesis. Systemic administration of non-refolded precipitated protein expressed in Escherichia coli caused growth regression of Lewis lung carcinoma, T241 fibrosarcoma, B16 melanoma and EOMA cells (O""Reilly et al. (1997) Cell 88:277-85)in a xenograft model. Moreover, no drug resistance was noted in three of the tumor types studied. Repeated cycles of administration with endostatin have been reported to result in tumor dormancy (Boehm et al.(1997) Nature 390:404-407).
The results from these angiostatin and endostatin studies open new avenues for treatment of cancer and provide promising routes for overcoming the drug resistance often seen during chemotherapy. However, in all of these investigations, a non-refolded precipitated form of the inhibitor protein was administered in the form of a suspension to tumor bearing animals. In addition, large amounts of protein were required to cause tumor regression and to lead to tumor dormancy. As pointed out by Kerbel ((1997) Nature 390:335-36), oral drug equivalents of these proteins are needed. Mechanistic investigations could be undertaken if recombinant forms of these proteins were available in soluble form. Moreover, initial testing could be done in vitro with soluble protein before studying its efficacy under in vivo conditions.
Furthermore, there have been reports that despite the great promise held by these proteins, evaluation of their clinical potential is stymied due to difficulties in producing enough of the protein to test, and inconsistent test results regarding their anti-angiogenic properties, e.g., anti-angiogenic activity (King, R. T., Wall Street J., Page 1, November 12 (1998); Leffe, D. N., BioWorld Today, 9:1, October 20 (1998)). There clearly exists at the present time a great need for a reproducible method of producing soluble forms of anti-angiogenic proteins with sufficient biological activity to be clinically effective and to reliably produce these proteins in high yields without sacrificing such critical activity.
The present invention relates to the discovery of a reproducible method of producing anti-angiogenic proteins with biological activity sufficient to be clinically effective. As described herein, the anti-angiogenic proteins encompassed by the present methods are reproducibly produced in high yields (for example from 10 to 20 mg/liter of culture medium. Importantly, the anti-angiogenic proteins produced by the methods described herein retain high biological activity.
Anti-angiogenic proteins are well-known to those of skill in the art. For example, angiostatin, endostatin, the 16 kD prolactin fragment, RNAisn, TNP470, 2-methoxy estradiol and heparin, to name a few. As used herein, the term xe2x80x9canti-angiogenic protein(s)xe2x80x9d encompass not only intact proteins, but mutants (e.g., with amino acid residues added, deleted or altered), fragments (e.g., specifically with amino acids deleted), derivatives (e.g., modified proteins or peptides, for example to reduce protease degradation) and fusion proteins wherein the fusion proteins comprise a combination of two or more known anti-angiogenic proteins (e.g., angiostatin and endostatin, or biologically active fragments of angiostatin and endostatin), or an anti-angiogenic protein in combination with a targeting agent (e.g., endostatin with epidermal growth factor (EGF) or RGD peptides), or an anti-angiogenic protein in combination with an immunoglobulin molecule (e.g., endostatin and IgG, specifically with the Fc portion removed). The term xe2x80x9cfusion proteinxe2x80x9d as used herein can also encompass additional components for e.g., delivering a chemotherapeutic agent, wherein a polynucleotide encoding the chemotherapeutic agent is linked to the polynucleotide encoding the anti-angiogenic protein. Fusion proteins can also encompass multimers of the anti-angiogenic protein, e.g., a dimer or trimer of endostatin.
It is also to be recognized that any of the anti-angiogenic proteins described herein can be post-translationally modified to encompass targeting moieties (e.g., vascular endotheleial growth factor (VEGF) or chemotherapeutic agents, such as ricin, or radioisotopes. Additional post-translational modifications can include multimerization, e.g., by chemical cross-linking using techniques well-know to those of skill in the art. However, for brevity, the term xe2x80x9canti-angiogenic proteinxe2x80x9d is used herein without specifically referring to mutant, derivatives, fragments and fusion proteins.
Encompassed by the present invention are methods of producing a biologically active anti-angiogenic protein, or a biologically active mutant, fragment, derivative or fusion protein thereof, using a prokaryotic or eukaryotic expression system. Specifically encompassed is the use of a yeast expression system, more specifically the Pichia pastoris yeast expression system.
The method steps involve inserting an isolated polynucleotide comprising a polynucleotide sequence encoding an anti-angiogenic protein, or mutant or derivative or fragment or fusion protein thereof, into a suitable expression vector e.g., prokaryotic or eukaryotic. Specifically encompassed by the present invention are yeast expression vectors. Suitable yeast expression vectors are well-known to those of skill in the art. A particularly suitable expression vector for use in the methods described herein is the commercially available Pichia vector comprising pPICzxcex1A plasmid wherein the plasmid contains a multiple cloning site. The polynucleotide sequences of anti-angiogenic proteins are well-known in the art, and novel anti-angiogenic protein sequences, as well as mutant sequences of known anti-angiogenic proteins are also described herein, and in PCT/US98/26058, xe2x80x9cRestin and Methods of Use Thereof,xe2x80x9d by Vikas P. Sukhatme, filed Dec. 8, 1998, and in U.S. Ser. No. 09/589,774, xe2x80x9cRestin and Methods of Use Thereofxe2x80x9d, by Vikas P. Sukhatme, filed Jun. 8, 2000, and in PCT/US98/26057, xe2x80x9cMutants of Endostatin, xe2x80x98EM1xe2x80x99 Having Anti-Angiogenic Activity and Methods of Use Thereof,xe2x80x9d by Vikas P. Sukhatme, filed Dec. 8, 1998, and in U.S. Ser. No. 09/589,887, xe2x80x9cAnti-Angiogenic Peptides and Method of Use Thereofxe2x80x9d, by Vikas P. Sukhatme, filed Jun. 8, 2000, the teachings of all of which are herein incorporated by reference in their entirety. Inserting the selected polynucleotide sequence into the vector is routine to those of skill, and is also described herein.
After inserting the selected polynucleotide into the vector, the vector is transformed into an appropriate yeast strain and the yeast strain is cultured (e.g., maintained) under suitable culture conditions for the production of the biologically active anti-angiogenic protein, thereby producing a biologically active anti-angiogenic protein, or mutant, derivative, fragment or fusion protein thereof. Typically the anti-angiogenic proteins are produced in quantities of about 10-20 milligrams, or more, per liter of culture fluid.
In one embodiment, the isolated polynucleotide encoding the anti-angiogenic protein additionally comprises a polynucleotide linker encoding a peptide. Such linkers are known to those of skill in the art and, for example, the linker can comprise at least one additional codon encoding at least one additional amino acid. Typically the linker comprises one to about twenty or thirty amino acids. Typically the linker is attached to the 5xe2x80x2 end of the polynucleotide encoding the anti-angiogenic protein, but can also be attached to the 3xe2x80x2 end. The polynucleotide linker is translated, as is the polynucleotide encoding the anti-angiogenic protein, resulting in the expression of an anti-angiogenic protein with at cast one additional amino acid residue at the amino or carboxyl terminus of the anti-angiogenic protein. For example, as described herein, the anti-angiogenic protein, endostatin is expressed using the methods described herein with two additional amino acid residues 5xe2x80x2 to endostatin. (See FIG. 5) These two additional amino acid residues are glutamic acid (E) and phinylalanine (F). Additionally, other amino acid residues can comprise the anti-antiogenic protein (See FIGS. 5 and 25). Importantly, the additional amino acid, or amino acids, do not compromise the activity of the anti-angiogenic protein. In fact, the anti-angiogenic proteins produced by the methods described herein exhibit superior biological activity to anti-angiogenic proteins produced by other expression methods. Typically, the concentrations of anti-angiogenic proteins produced in this embodiment are about 10-20 milligrams per liter of culture medium.
In another embodiment of the present invention, the eukaryotic vector comprises a yeast vector comprising a histadine tag motif. As described herein, one method uses a pPICzxcex1A plasmid wherein the plasmid contains a multiple cloning site which contains a His.Tag motif (also referred to herein as pPICzxcex1A/HIS.). Additionally the vector can be modified to add a Ndel site, or other suitable restriction sites. Such sites are well known to those of skill in the art. Anti-angiogenic proteins produced by this embodiment comprise a histidine tag motif (His.tag) comprising one, or more histadine, typically about 5-20 histidines. Surprisingly, this His.tag does not compromise anti-angiogenic activity. In fact, the anti-angiogenic proteins produced by the methods described herein exhibit superior biological activity to anti-angiogenic proteins produced by other expression methods. Again, the biologically active protein is typically produced at concentrations of about 10-20 milligrams per liter of culture medium (fluid).
Combinations of the above embodiments are also encompassed by the present invention. For example, the selected polynucleotide can comprise a linker and be inserted into a vector comprising his.Tag motif. (See FIGS. 5 and 25).
Also encompassed by the present invention are the anti-angiogenic proteins produced by the methods described herein. Surprisingly, the proteins produced by the methods described herein are produced in high yields and have biological activity sufficient for testing the clinical efficacy of these proteins to inhibit (completely, or substantially reduce) unwanted angiogenic activity.
Further encompassed by this invention are methods of using the anti-angiogenic proteins produced by the methods described herein. For example, the anti-angiogenic proteins described herein can be used to test the efficacy of treating human malignant tumors wherein such treatment would result in the regression, partial, or complete, of the tumor. These proteins can also be used to treat other diseases that have undesirable angiogenesis as described herein.
Further encompassed by the present invention are compositions comprising an effective amount of an anti-angiogenic protein produced by the methods described herein, and a pharmaceutically acceptable carrier. An effective amount of an anti-angiogenic protein is described herein, and typically is an amount sufficient to inhibit endothelial activity such as endothelial cell migration, inhibition of tumor growth, arrest of endothelial cells in G1 phase of the cell cycle, and inducing apoptosis in endothelial cells. Assays to determine these activities are described herein. xe2x80x9cED50xe2x80x9d is an abbreviation for the amount of anti-angiogenic protein which reduces a biological effect by one-half relative to the biological effect seen in the absence of the anti-angiogenic protein. Comparing the concentration of an anti-angiogenic protein required to reduce a biological effect by one-half, in for example, the endothelial cell proliferation assay, is a useful measure of biological activity and allows comparison between compositions.
Also encompassed by the present invention are methods of using the anti-angiogenic proteins described herein to inhibit undesirable angiogenic activity. The methods encompassed by the present invention can inhibit endothelial cell migration, inhibit tumor growth in mammals, arrest endothelial cells in G1 phase of the cell cycle, and induce apoptosis in endothelial cells. The anti-angiogenic proteins of the present invention specifically and reversibly inhibit endothelial cell proliferation. The inhibitor protein molecules of the invention are also useful as a birth control drug, and for treating other angiogenesis-related diseases, particularly angiogenesis-dependent cancers and tumors.
As a result of the invention described herein, quantities of anti-angiogenic proteins, mutants, derivatives and fragments thereof, as well as anti-angiogenic fusion proteins are now available with sufficient biological activity for the study and treatment of angiogenic diseases. The unexpected and surprising ability of these anti-angiogenic compounds to treat and alleviate angiogenesis-dependent cancers and tumors answers a long felt unfulfilled need in the medical arts, and provides an important benefit to mankind.